Labeled compounds incorporating stable isotopes have been used in the analysis of metabolic pathways and human nutrition, and are becoming increasingly important in disease diagnosis. In isotopic labeling, one or more of the atoms of a molecule of interest are substituted for an atom of the same element, but of a different isotope. Because the atom has the same number of protons, it will behave the same chemically as other atoms in the compound. However, the difference in the number of neutrons imparts a mass difference that can be detected separately from the other atoms of the same element.
Isotopically altered compounds can be easily identified through various techniques, such as nuclear magnetic resonance (NMR), mass spectrometry (MS), or autoradiography. By using isotopically labeled compounds, the metabolic and physical processes occurring in an organism can be studied by observing the path that labeled material takes and the types of metabolic end products that the label is eventually found in. Isotopic labels can be generally divided into two classes; those that are radioactive, such as carbon-14, tritium, and technetium-99 m, and those that are stable, such as carbon-13, nitrogen-15, and oxygen-18. While radioactive labeled compounds can be identified by a greater number of techniques, their use typically requires specialized nuclear medicine facilities and may be contra-indicated in children and women of child-bearing age, thus limiting their usefulness.
Carbon-13 [13C] is a particularly useful isotope, as carbon is present in essentially all organic material and 13C is a non-radioactive isotope that is readily identified. In 13C analysis, 13C is introduced into one or more functional groups in a substrate. The functional groups are linked to the rest of the molecule through bonds that are cleaved by specific enzymes. Once cleavage occurs, the functional group is typically further oxidized until 13CO2 is produced, which is then excreted in the breath. The appearance of excess 13CO2 in respiration can be used to indicate the presence and amount of enzymatic activity or indicate the presence of a foreign substance such as bacteria. This use of 13C has led to the development of a number of 13C-based breath tests. See Peter D. Klein, “13C Breath Tests: Visions and Realities”, Journal of Nutrition, 131, 1637S, 2001, the disclosure of which is incorporated herein by reference.
To conduct breath tests, or other tests using 13C label, a digestible source incorporating the labeled material is often needed. 13C labeled bicarbonate ingested by a subject in a breath study to measure gastric emptying produced unreliable results when administered without being incorporated into a digestible material. 13C label incorporated into microorganisms has proven to be a superior vector for introducing labeled material to a subject for breath tests.
Algae are microorganisms that have been found to be useful as a digestible source in breath tests. Algae labeled in this fashion have been defined as a drug by the FDA as have previous substrates for other legally marketed 13C-labeled breath tests. The proteins, lipids, and carbohydrates of these organisms can be engineered to contain high levels of 13C label. If such organisms are used, they can be readily introduced for diagnostic and physiological measurements by incorporating them in an edible product such as biscuits. See U.S. Pat. No. 5,707,602, the disclosure of which is incorporated herein by reference, for an example of this approach.
Various single-celled organisms have been cultivated in the presence of a 13C source in order to provide labeled organisms. To increase the incorporation of 13C, as opposed to atmospheric 12C, cultivation of these organisms is typically conducted in a bioreactor, an apparatus that provides the conditions necessary for growth while preventing contamination. Algae are useful organisms for providing 13C labeled biomass, as the nutrient requirements of algae are relatively inexpensive. To culture algae, it is necessary to provide a carbon source, which is typically CO2 gas, various trace nutrients, and light, which the algae use to drive photosynthesis.
As CO2 gas is the traditional carbon source for cultivating algal biomass, previous efforts to produce 13C labeled algae have used 13CO2 gas as the carbon source. 13CO2 gas, however, is relatively expensive and a significant amount may be lost through waste. Also, typically 13CO2 gas is bubbled into a growth chamber of a bioreactor. If fittings, seals, and other chamber components fail during the process, 13C label may be diluted and there is an increased chance of contamination. Even when fittings and seals remain sealed, a significant amount of the 13CO2 gas may simply pass through the system without being absorbed into the biomass. Furthermore, while gas volumes can be calculated theoretically, it is difficult to reliably administer precise volumes of gas, such as 13CO2. Finally, use of 13CO2 gas as the carbon source requires the use of bulky pressurized cylinders that are awkward to transport and handle.
A method of consistently producing a uniformly 13C labeled biomass without the use of 13CO2 gas and its associated contamination, wastage, and handling problems, for use as component of a diagnostic test kit or as an active pharmaceutical ingredient in a drug product would be desirable.